Protein-protein interactions are central to biological systems. Methods (2-hydrid or affinity isolation) for the systematic identification of protein-protein interaction networks have been applied to a number of model organisms (e. g. yeast, C. elegans, E. coli, etc.). The ever increasing number of available complete genome sequences for non-model bacteria has led to the need for additional methods for system wide functional genomic analysis. To date, however, interactome mapping has been completed exclusively on model organisms such as E. coli and Saccharomyces cerevisiae. We have developed methodology for determining protein-protein interaction networks in the non-model gamma-proteobacterium Shewanella oneidensis. This involves both endogenous and exogenous affinity isolation of protein complexes using fusion protein baits bearing tandem affinity tags followed by identification of interactors using mass spectrometry.

Initial experiments have focused on a number of protein complexes conserved across multiple microbial species to validate this approach for mapping microbial interactomes. The results of mapping the interaction subnetworks for homologs of open reading frames annotated to be part of DNA-directed RNA polymerase, F1F0-ATP synthase, the degradosome and others have been analyzed for S. oneidensis. Results will be compared to those obtained for homologous systems determined in other organisms (e.g. E.coli) available in public protein interaction databases (BIND, DIP, Prolinks, etc). Our data indicate that endogenous and exogenous pulldown approaches provide complementary information about protein – protein interactions and demonstrate the feasibility of our high-throughput approaches for mapping microbial interaction networks.

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